Acyl amino acid-releasing enzyme

When the enzyme is used to catalyse the synthesis of a peptide bond, the solvent is either non-aqueous or contains only a low concentration of water. In addition, of course, an amino component such as an amino acid or peptide ester replaces the water in the second step. Obviously, the amino component must be unprotonated for reaction to succeed. Synthesis is favoured over hydrolysis of the resultant peptide because an amide is kinetically a much worse substrate for a proteinase than is an ester. The rapid acylation of a proteinase by an TV-protected amino acid or peptide aryl ester can be demonstrated experimentally using a stopped-flow apparatus with spectrophotometric facilities. A rapid burst of phenol is followed by steady-state release, showing that acylation of the enzyme is faster than hydrolysis of the acy-lated enzyme. No such burst is detectable if, for example, an TV-acylated amino acid anilide is used as substrate. In fact, acylation is the rate-determining step with amide substrates. 

Another possible mechanism for the racemization of amino acid esters involves the in situ, transient, formation of Schiff s bases by reaction of the amine group of an amino acid ester with an aldehyde. Using this approach, DKR of the methyl esters of proline 5 and pipecolic acid 6 was achieved using lipase A from C. ant-arclica as the enantioselective hydrolytic enzyme and acetaldehyde as the racemiz-ing agent (Scheme 2.4). Interestingly, the acetaldehyde was released in situ from vinyl butanoate, which acted as the acyl donor, in the presence of triethylamine. The use of other reaction additives was also investigated. Yields of up to 97% and up to 97% e.e. were obtained [6]. 

The enzyme works optimally at pH > 9 with N-protected amino acid esters as acyl donors, and amino acids or amino acid amides as nucleophiles (acceptors). At that pH value it has high esterase activity an amino acyl-enzyme complex will be formed rapidly which transfers the acyl residue to the acceptor molecule (transpeptidation), and since at alkaline pH the rate of peptide cleaving is minimal, the product formed will be released in excellent yield. As an example the last coupling step in a synthesis of the opioide peptide Met-enkephalin that started with benzoylarginine ethylester may be shown (Fig. 8). 

In each cell synthesizing proteins, there are at least 20 different amino acyl RNA synthetases, one for each amino acid. The overall reaction catalyzed by the synthetase leads to the formation of an ester between amino acid and tRNA. Thus, the reaction takes place in two major steps a first, already discussed, in which the amino acid is activated and complexed with the enzyme, and a second, in which the activated amino acid is transferred to tRNA. ATP and Mg are required for the first step. In the first step, the enzyme catalyzes the formation of a mixed anhydride between the carboxyl of the amino acid and the 5 -P04 of AMP. Pyrophosphate is released in the course of the reaction. The anhydride never appears as a free intermediate but remains tightly attached to the enzyme. 

The only requirement for design of an aza-peptide reagent for a new serine protease is knowledge of the enzyme s substrate specificity so that an appropriate side chain may be placed on the aza-amino acid residue. The employment of p-nitrophenol as a product of the acylation step is advantageous since it is a good leaving group, and the release of p-nitro-... 

Amino acids bind to activating enzymes (amino acyl-tRNA synthetases), which recognize both the amino acid and the appropriate tRNA molecule. The first step is reaction between the amino acid and ATP, to form amino acyl AMP, releasing pyrophosphate. The amino acyl AMP then reacts with the -CCA tail of tRNA to form amino acyl-tRNA, releasing AMP... 

---